Cadaveric evaluation of sternal reconstruction using the pectoralis muscle flap.

BACKGROUND: Deep sternal wound infection is a significant complication of open cardiac surgery associated with increased mortality and morbidity. The use of muscle flaps, such as the pectoralis major advancement flap, in deep sternal wound infection reconstruction reduces hospital stay and mortality. However, the lower end of the sternum is remote from the vascular supply and cover is therefore problematic in many cases. METHODS: This study aimed to determine the distance (cm) and surface area (cm2 ) of sternum covered when the pectoralis major muscle is sequentially dissected from the sternocostal origin and humeral insertion using 10 cadaveric specimens. RESULTS: The largest proportion of sternum was covered when both the origin and insertion were divided, allowing the flap to be islanded on its vascular pedicle. There was a statistically significant difference when the pectoralis major was divided from the origin and insertion compared to division of the origin alone (P < 0.01). The average area covered with sternocostal origin division alone was 55.43 cm2 compared to 85.36 cm2 after division of both the origin and insertion. CONCLUSION: Division of both the sternocostal origin and humeral insertion of the pectoralis major muscle represents an effective means to increase sternal coverage. This study describes the average distance and area covered by sliding pectoralis major muscle advancement flaps. These measurements could better inform plastic surgeons when evaluating reconstructive options in sternal defects.

Changes in dermal matrix in the absence of Rac1 in keratinocytes.

Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed that the deletion of Rac1 in keratinocytes causes heightened inflammation due to aberrant crosstalk with immune cells. Indeed, the skin of these mice shows a higher inflammatory response to the induction of irritant contact dermatitis (ICD), and also even to treatment with a vehicle alone, compared with controls. As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction of ICD on the tissue localisation and arrangements of dermal collagen. Qualitative analysis did not reveal evidence for the formation of pathologies in the dermis. However, quantitative analysis did reveal some perturbations in the dermal matrix, namely that only the combination of the lack of Rac1 and ICD affects the architectural organisation of the dermal collagen, and that a higher inflammatory state in the tissue (i.e. when Rac1 is deleted in the keratinocytes or ICD is induced in the skin, or a combination of both) influences the diameter of the collagen fibrils. It is proposed that this increase in the diameter of collagen fibrils due to inflammation may serve as pre-fibrotic marker enabling earlier determination of fibrosis and earlier treatment. This study has revealed previously unknown effects on the ECM due to the deletion of Rac1 in keratinocytes.

Effects of Polydopamine Functionalization on Boron Nitride Nanotube Dispersion and Cytocompatibility.

Boron nitride nanotubes (BNNTs) have unique physical properties, of value in biomedical applications; however, their dispersion and functionalization represent a critical challenge in their successful employment as biomaterials. In the present study, we report a process for the efficient disentanglement of BNNTs via a dual surfactant/polydopamine (PD) process. High-resolution transmission electron microscopy (HR-TEM) indicated that individual BNNTs become coated with a uniform PD nanocoating, which significantly enhanced dispersion of BNNTs in aqueous solutions. Furthermore, the cytocompatibility of PD-coated BNNTs was assessed in vitro with cultured human osteoblasts (HOBs) at concentrations of 1, 10, and 30 µg/mL and over three time-points (24, 48, and 72 h). In this study it was demonstrated that PD-functionalized BNNTs become individually localized within the cytoplasm by endosomal escape and that concentrations of up to 30 µg/mL of PD-BNNTs were cytocompatible in HOBs cells following 72 h of exposure.

NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and Rho GTPases in cell migration.

SIGNIFICANCE: Rho GTPases are historically known to be central regulators of actin cytoskeleton reorganization. This affects many processes including cell migration. In addition, members of the Rac subfamily are known to be involved in reactive oxygen species (ROS) production through the regulation of NADPH oxidase (Nox) activity. This review focuses on relationships between Nox-regulated ROS, Rho GTPases, and cytoskeletal reorganization, in the context of cell migration. RECENT ADVANCES: It has become clear that ROS participate in the regulation of certain Rho GTPase family members, thus mediating cytoskeletal reorganization. CRITICAL ISSUES: The role of the actin cytoskeleton in providing a scaffold for components of the Nox complex needs to be examined in the light of these new advances. During cell migration, Rho GTPases, ROS, and cytoskeletal organization appear to function as a complex regulatory network. However, more work is needed to fully elucidate the interactions between these factors and their potential in vivo importance. FUTURE DIRECTIONS: Ultrastructural analysis, that is, electron microscopy, particularly immunogold labeling, will enable direct visualization of subcellular compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location, these data will contribute significantly to our understanding of this intricate network under physiological conditions. Based on this, in vivo and in vitro studies can then be combined to elucidate the signaling pathways involved and their targets.

RAC1 in keratinocytes regulates crosstalk to immune cells by Arp2/3-dependent control of STAT1.

Crosstalk between keratinocytes and immune cells is crucial for the immunological barrier function of the skin, and aberrant crosstalk contributes to inflammatory skin diseases. Using mice with a keratinocyte-restricted deletion of the RAC1 gene we found that RAC1 in keratinocytes plays an important role in modulating the interferon (IFN) response in skin. These RAC1 mutant mice showed increased sensitivity in an irritant contact dermatitis model, abnormal keratinocyte differentiation, and increased expression of immune response genes including the IFN signal transducer STAT1. Loss of RAC1 in keratinocytes decreased actin polymerization in vivo and in vitro and caused Arp2/3-dependent expression of STAT1, increased interferon sensitivity and upregulation of aberrant keratinocyte differentiation markers. This can be inhibited by the AP-1 inhibitor tanshinone IIA. Loss of RAC1 makes keratinocytes hypersensitive to inflammatory stimuli both in vitro and in vivo, suggesting a major role for RAC1 in regulating the crosstalk between the epidermis and the immune system.

Rho GTPases and Nox dependent ROS production in skin. Is there a connection?

Rho GTPases are a family of small GTP binding proteins most commonly known for the regulation of many cellular processes, including actin cytoskeleton re-organisation, cell proliferation, signal transduction and regulation of apoptosis. Additionally, a link between Rho GTPases and reactive oxygen species (ROS) has been shown. In line with the growing interest in the role of ROS in cell biology, the relevance of this connection is becoming increasingly clearer. ROS production is classically associated with oxidative metabolic pathways (e.g. respiratory chain, arachidonic acid). During these metabolic pathways, ROS are produced as by-products and these can be potentially toxic. However, numerous cell types contain dedicated enzymatic complexes, i.e., NADPH oxidase (Nox) complexes, for regulated production of ROS. This regulated production of ROS seems to be important for a number of fundamental cell biological processes, including cell growth, differentiation, migration, angiogenesis, aimed at maintaining tissue homeostasis. Data suggests that skin cells are capable of a regulated ROS production via Nox complexes. Members of the Rho GTPase family have been found to play a central regulatory role in Nox activity. In the present review we will focus on the involvement of Rho GTPases in regulated production of ROS with special emphasis on the skin. We will also discuss the possibility that some in vivo effects of the deletion of members of the Rho GTPase family in skin cells could potentially be linked to a reduced ability of regulated ROS production.

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGF{beta} pathways.

N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGFbeta pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGFbeta target gene. Inhibition of TGFbeta signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGFbeta signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGFbeta pathway in keratinocytes in vivo and in vitro.

A non-apoptotic role for caspase-9 in muscle differentiation.

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.